PROJECT SUMMARY Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, is well adapted to its multiple tick and mammalian hosts and, despite a minimalist genome leaving it devoid of many functions, has evolved highly effective mechanisms for evading different host defenses. As a part of survival within such disparate hosts, one common requirement is the ability to neutralize oxidative and nitrosative stress responses. B. burgdorferi have a striking dearth of reactive oxygen species (ROS) and reactive nitrogen species (RNS) detoxification enzymes seen in most bacterial pathogens suggesting that it uses novel mechanisms to evade these defenses. In our preliminary data, we have identified over 30 new genes that appear to be involved in B. burgdorferi resistance to ROS or RNS. Genes were identified by exposure of a transposon library to either ROS or RNS stress followed by sequencing (Tn-seq) to precisely determine the fitness of individual mutants. Identified genes were confirmed using individual mutants and complemented mutants. Mutants in genes identified as having a role in ROS and/or RNS resistance were tested for their infectivity into their natural tick and mouse hosts. We identified multiple mutants with defects in infectivity into mice or ticks. In this proposal, we seek to understand the unique mechanisms used by B. burgdorferi to evade oxidative and nitrosative killing. We are proposing the following 3 Specific Aims. First, in Aim 1, we will assess the role of ROS/RNS responsive proteins of B. burgdorferi for survival during mammalian infection. Using both mutant bacteria and knockout mice devoid of specific components of the ROS/RNS defenses, we will dissect the role of specific genes at different time points in the infectious cycle. We will use in vivo imaging, RNA-seq and quantitative rt-PCR to pinpoint the role of individual genes. Next, in Aim 2, we will assess the role of ROS/RNS responsive proteins of B. burgdorferi for survival in its tick host using similar strategies to Aim 1. Finally, in Aim 3, we will determine how the candidate proteins mediate resistance to ROS. In this Aim we will test the predicted activity of several targeted proteins for their role in detoxification of ROS/RNS. This study represents the first genetic screen to globally assess how B. burgdorferi combats ROS/RNS. Given the importance of this response in host innate immunity, the characterization of these borrelial genes/proteins will provide important insight into the ability of B. burgdorferi to avoid clearance and persist for prolonged periods within the hosts they occupy in nature. Insights into mechanisms by which the organism is able to escape immune mediated killing are likely to have important implications for understanding human Lyme disease.